权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Development of gene therapy for dystrophic epidemolysis bullosa with artificial adhension molecule

人工粘附分子治疗营养不良性大疱性流行病溶解症基因治疗的进展

基本信息

批准号：
14370257
负责人：
TAMAI Katsuto
金额：
$ 8.9万
依托单位：
Osaka University (2003)Hirosaki University (2002)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

项目摘要

Because of type VII collagen gene (COL7A1) defect, immune tolerance for type VII collagen must be disrupted in the patients of dystrophic epidermolysis bullosa (EB). However, since many EB patients express abnormal type VII collagen truncated after NC-1 domain which function to bind to the basement membrane, we have constructed expression vector of the artificial molecule composed of NC-1 domain of type VII collagen and fibronectin collagen binding domain chimera (NC-1/FNCBD) to expect adhesive activity at cutaneous basement membrane region and immune tolerance of the chimera molecule. Transfection of the NC-1/FNCBD expression vector in HaCat keratinocytes provided efficient expression of the chimera molecule both in the cells and the culture medium. This artificial peptide may function to rescue severe blistering phenotype of the dystrophic EB patients.To introduce the NC-1/FNCBD expression vector to the living skin, novel technology must be developed to introduce high molecular weight molecule, such as plasmid DNA, to the skin, as barrier function of the skin is strong enough to prohibit penetration of the molecule bigger than 1 kDa in size. We applied chemical pealing and ultrasonic sound (US) energy to introduce plasmid DNA to the skin. 50% glycolic acid (GA) treatment for 10 minutes was enough to break horny layer barrier function, and application of US energy on the GA-treated skin enabled introduction of the NC-1/FNCBD expression vector to the skin soaked in the plasmid solution, resulting in the expression of the NC-1/FNCBD molecule at dermo-epidernal junction.

由于VII型胶原基因（COL 7A 1）缺陷，营养不良性大疱性表皮病（EB）患者对VII型胶原的免疫耐受必须被破坏。然而，由于许多EB患者表达在NC-1结构域之后截短的异常VII型胶原，其功能是结合基底膜，因此我们构建了由VII型胶原的NC-1结构域和纤连蛋白胶原结合结构域嵌合体（NC-1/FNCBD）组成的人工分子的表达载体，以期望嵌合体分子在皮肤基底膜区域的粘附活性和免疫耐受。NC-1/FNCBD表达载体在HaCat角质形成细胞中的转染提供了嵌合体分子在细胞和培养基中的有效表达。为了将NC-1/FNCBD表达载体导入活的皮肤，必须开发新的技术来将高分子量分子如质粒DNA导入皮肤，因为皮肤的屏障功能足够强以阻止尺寸大于1 kDa的分子的渗透。我们应用化学剥离和超声波（US）能量将质粒DNA引入皮肤。50%乙醇酸（GA）处理10分钟足以破坏角质层屏障功能，并且在GA处理的皮肤上施加US能量能够将NC-1/FNCBD表达载体引入到浸泡在质粒溶液中的皮肤中，导致NC-1/FNCBD分子在真皮-表皮接合处表达。